Substantially pure reverse transcriptases and methods of production thereof

ABSTRACT

The present invention provides substantially pure reverse transcriptases, which are preferably substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in synthesizing, amplifying or sequencing nucleic acid molecules including through the use of the polymerase chain reaction, particularly RT-PCR.

FIELD OF THE INVENTION

[0001] The present invention is in the fields of molecular biology,protein chemistry and protein purification. Specifically, the inventionprovides compositions comprising reverse transcriptases (RTs) andmethods for the production of such reverse transcriptase enzymes. Suchmethods provide for reverse transcriptases that are substantially freefrom contamination by nucleic acids and other unwanted materials orproteins. Compositions comprising the reverse transcriptase enzymes ofthe present invention may be used in a variety of applications,including synthesis, amplification and sequencing of nucleic acids.

BACKGROUND AND SUMMARY OF THE INVENTION

[0002] A variety of techniques may be employed to facilitate thepreparation of intracellular proteins from microorganisms. Typically,the initial steps in these techniques involve lysis or rupture of thebacterial cells, to disrupt the bacterial cell wall and allow release ofthe intracellular proteins into the extracellular milieu. Following thisrelease, the desired proteins are purified from the extracts, typicallyby a series of chromatographic steps.

[0003] Several approaches have proven useful in accomplishing therelease of intracellular proteins from bacterial cells. Included amongthese are the use of chemical lysis, physical methods of disruption, ora combination of chemical and physical approaches (reviewed in Felix,H., Anal. Biochem. 120:211-234 (1982)).

[0004] Chemical methods of disruption of the bacterial cell wall thathave proven useful include treatment of cells with organic solvents suchas toluene (Putnam, S. L., and Koch, A. L., Anal. Biochem. 63:350-360(1975); Laurent, S. J., and Vannier, F. S., Biochimie 59:747-750 (1977);Felix, H., Anal. Biochem. 120:211-234 (1982)), with chaeotropes such asguanidine salts (Hettwer, D., and Wang, H., Biotechnol. Bioeng.33:886-895 (1989)), with antibiotics such as polymyxin B (Schupp, J. M.,et al., BioTechniques 19:18-20 (1995); Felix, H., Anal. Biochem.120:211-234 (1982)), or with enzymes such as lysozyme or lysostaphin(McHenty, C. S., and Kornberg, A., J. Biol. Chem. 252(18):6478-6484(1977); Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990);Hughes, A. J., Jr., et al., J. Cell Biochem. Suppl. 016 (Part B):84(1992); Sambrook, J., et al., in Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, p. 17-38 (1989); Ausubel, F. M., et al., in Current Protocols inMolecular Biology, New York: John Wiley & Sons, p. 4.4.1-4.47 (1993)).The effects of these various chemical agents may be enhanced byconcurrently treating the bacterial cells with detergents such as TritonX-100®, sodium dodecylsulfate (SDS) or Brij 35 (Laurent, S. J., andVannier, F. S., Biochimie 59:747-750 (1977); Felix, H., Anal. Biochem.120:211-234 (1982); Hettwer, D., and Wang, H., Biotechnol. Bioeng,33:886-895 (1989); Cull, M., and McHenry, C. S., Meth. Enzymol.182:147-153 (1990); Schupp, J. M., et al., BioTechniques 19:18-20(1995)), or with proteins or protamines such as bovine serum albumin orspermidine (McHenry, C. H. and Kornberg, A., J. Biol. Chem. 252(18):6478-6484 (1977); Felix, H., Anal. Biochem. 120:211-234 (1982); Hughes,A. J., Jr., et al., J. Cell Biochem. Suppl. 0 16 (Part B):84 (1992)).

[0005] In addition to these various chemical treatments a number ofphysical methods of disruption have been used. These physical methodsinclude osmotic shock, e.g., suspension of the cells in a hypotonicsolution in the presence or absence of emulsifiers (Roberts, J. D., andLieberman, M. W., Biochemistry 18:4499-4505 (1979); Felix, H., Anal.Biochem. 120:211-234 (1982)), drying (Mowshowitz, D. B., Anal. Biochem.70:94-99 (1976)), bead agitation such as ball milling (Felix, H., Anal.Biochem. 120:211-234 (1982); Cull, M., and McHenry, C. S., Meth.Enzymol. 182:182:147-153 (1990)), temperature shock, e.g., freeze-thawcycling (Lazzarini, R. A., and Johnson L. D., Nature New Biol. 243:17-20(1975); Felix, H., Anal, Biochem. 120:211-234 (1982)), sonication (Amos,H., et al., J. Bacteriol. 94:232-240 (1967); Ausubel, F. M., et al., inCurrent Protocols in Molecular Biology, New York:John Wiley & Sons, pp.4.4.1-4.47 (1993)) and pressure disruption, e.g., use of a frenchpressure cell (Ausubel, F. M., et al., in Current Protocols in MolecularBiology, New York:John Wiley & Sons, pp. 16.8.6-16.8.8 (1993)). Otherapproaches combine these chemical and physical methods of disruption,such as lysozyme treatment followed by sonication or pressure treatment,to maximize cell disruption and protein release (Ausubel, F. M., et al.,in Current Protocols in Molecular Biology, New York:John Wiley & Sons,pp. 4.4.1-4.47 (1993)).

[0006] These disruption approaches have several advantages, includingtheir ability to rapidly and completely (in the case of physicalmethods) disrupt the bacterial cell such that the release ofintracellular proteins is maximized. In fact, these approaches have beenused in the initial steps of processes for the purification of a varietyof bacterial cytosolic enzymes, including natural and recombinantproteins from mesophilic organisms such as Escherichia coli, Bacillussubtilis and Staphylococcus aureus (Laurent, S. J., and Vannier, F. S.,Biochimie 59:747-750 (1977); Cull, M., and McHenry, C. S., Meth.Enzymol. 182:147-153 (1990); Hughes, A. J., Jr., et al., J. CellBiochem. Suppl. 0 16 (Part B):84 (1992); Ausubel, F. M., et al., inCurrent Protocols in Molecular Biology, New York: John Wiley & Sons, pp.4.4.1-4.47 (1993)), as well as phosphatases, restriction enzymes, DNA orRNA polymerases and other proteins from thermophilic bacteria andarchaea.

[0007] However, these methods possess distinct disadvantages as well.For example, the physical methods by definition involve shearing andfracturing of the bacterial cell walls and plasma membranes. Theseprocesses thus result in extracts containing large amounts ofparticulate matter, such as membrane or cell wall fragments, which mustbe removed from the extracts, typically by centrifugation, prior topurification of the enzymes. This need for centrifugation limits thebatch size capable of being processed in a single preparation to that ofavailable centrifuge space; thus, large production-scale preparationsare impracticable if not impossible. Furthermore, physical methods, andmany chemical techniques, typically result in the release from the cellsnot only of the desired intracellular proteins, but also of undesirednucleic acids and membrane lipids (the latter particularly resultingwhen organic solvents are used). These undesirable cellular componentsalso complicate the subsequent processes for purification of the desiredproteins, as they increase the viscosity of the extracts (Sambrook, J.,et al., in: Molecular Cloning: A Laboratory Manual, 2^(nd) ed., ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 17-38(1989); Cull, M., and McHenry, C. S., Meth. Enzynol. 182:147-153(1990)), and bind with high avidity and affinity to nucleic acid-bindingproteins such as DNA polymerases, RNA polymerases and restrictionenzymes.

[0008] One problem associated with these approaches is that the enzymepreparations are typically contaminated with nucleic acids (e.g., RNAand DNA). This contaminating nucleic acid may come not only from theorganisms which are the source of the enzyme, but also from unknownorganisms present in the reagents and materials used to purify theenzyme after its release from the cells. Since reverse transcriptaseenzymes are routinely used in techniques of amplification and synthesisof nucleic acid molecules (e.g., the Polymerase Chain Reaction (PCR),particularly RT-PCR) the presence of contaminating DNA or RNA in theenzyme preparations is a significant problem since it can give rise tospurious amplification or synthesis results. Thus, a need exists forpreparation of reverse transcriptase enzymes that are substantially freeof contamination by nucleic acids.

[0009] Instead of attempting to remove nucleic acids from preparationsof reverse transcriptase enzymes, a more reasonable and successfulapproach would be to prevent contamination of the enzymes by nucleicacids from the outset in the purification process. Such an approachwould be two-pronged: 1) preventing release of nucleic acids from thebacterial cells during permeabilization of the cells to release theenzymes; and 2) preventing contamination of the enzymes during thepurification process itself. Furthermore, an optimal method wouldobviate the need for centrifugation in the process, thus allowinglarge-scale, and even continuous, production of nucleic acid-freereverse transcriptase enzymes. The present invention provides suchmethods, and reverse transcriptase enzymes produced by these methods.

[0010] The present invention generally provides methods of making areverse transcriptase enzyme comprising permeabilizing a cellular sourceof reverse transcriptase (e.g., bacterial cells) to form spheroplasts orprotoplasts and isolating the reverse transcriptase enzyme. Preferably,the methods are conducted under conditions favoring the partitioning ofnucleic acids from the reverse transcriptase enzyme. In particular, theinvention relates to a method for isolation or purification of reversetranscriptases comprising cell permeabilization, filtration andisolation.

[0011] The invention is particularly directed to methods wherein thepermeabilization of the cells is accomplished by contacting the cellswith an aqueous solution comprising at least one of: a chaeotropicagent, preferably a guanidine salt and most preferably guanidinehydrochloride; and/or a nonionic detergent, preferably Triton X-100and/or sodium deoxycholic acid. The invention is further directed tosuch methods wherein the conditions favoring the partitioning of nucleicacids from the reverse transcriptase enzyme comprise formation of anfiltrate (e.g., ultrafiltrate) by filtration (e.g., microfiltration) ofthe cellular source subjected to permeabilization (particularly of thespheroplasts or protoplasts) through a semi-permeable membrane, which ispreferably a hydrophilic dialysis membrane, preferably in the presenceof a salt, preferably ammonium sulfate, and purification or isolation ofthe reverse transcriptase enzyme from the filtrate, preferably bychromatography using sterile materials. The invention is particularlydirected to such methods wherein bacterial cells providing the reversetranscriptase enzyme are used, preferably prokaryotic cells such asthose of species of the genera Escherichia (preferably E. coli),Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus,Clostridium, Chlamydia, Neisseria, Treponema, Klebsiella, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Xanthomonas and Streptomyces. In anotheraspect, the cellular source of reverse transcription is a recombinantcellular source.

[0012] The invention also provides the reverse transcriptase enzymes, ormutants, derivatives or fragments thereof, that are made according tothe methods provided. The invention is also directed to methods foramplifying or synthesizing a nucleic acid molecule comprising contactinga nucleic acid molecule (e.g., template) with an reverse transcriptasemade according to the methods of the present invention under conditionsto make a first nucleic acid molecule complementary to all or a portionof the template. Such synthesis or amplification may further compriseincubating the reaction with one or more polymerases (DNA polymerases,preferably thermostable DNA polymerases such as Tne, Tma, Taq etc. ormutants, derivatives or fragments thereof) under conditions sufficientto make a second nucleic acid molecule complementary to all or a portionof the first nucleic acid molecule.

[0013] The invention also provides kits for amplifying or synthesizingnucleic acid molecules comprising a carrier means having in closeconfinement therein one or more container means, wherein said kit maycomprise at least one component selected from one or more reversetranscriptases produced according to the invention, one or morepolymerases (e.g., DNA polymerases), one or more nucleotides orderivatives thereof, one or more primers, and one or more synthesis oramplification reaction buffers.

[0014] Other features and advantages of the present invention will beapparent to those skilled in the art from the following description ofthe preferred embodiments and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Overview

[0016] The present invention in a preferred aspect provides reversetranscription enzymes that are substantially pure and more preferablyreverse transcriptases that are substantially free of nucleic acids. Asused herein, the term “substantially free of nucleic acids” means anenzyme composition that comprises no nucleic acids, or that comprisesnucleic acids below the level of detection, when assayed by standardbiochemical assays for nucleic acids. Such assays may include gelelectrophoresis (e.g., agarose gel electrophoresis coupled with nucleicacid staining such as ethidium bromide, acridine orange or Hoechststaining), spectrophotometry (e.g., ultraviolet, atomic absorption, NMRor mass spectrometry), chromatography (liquid, gas, HPLC or FPLC), or byfunctional assays for nucleic acids detection such as amplification. Anexample of such functional assay is based on measuring incorporation oflabeled nucleotides (e.g., radio labeled, enzyme labels,chemiluminescent labels, etc.) by the enzyme preparation in a“no-template” nucleic acid amplification reaction. These biochemical andfunctional assays are described in more detail below and in Example 3.The invention also provides methods for the production of these enzymes,and compositions and kits comprising these enzymes which may be used insynthesis or amplifying nucleic acid molecules, including through use ofthe polymerase chain reaction (PCR).

[0017] Briefly summarized, the present invention utilizes a schemecomprising permeabilization of cells (preferably bacterial cells) toform spheroplasts or protoplasts, filtration (e.g., microfiltration) ofthe spheroplasts or protoplasts to form a filtrate (e.g.,microfiltrate), ultrafiltration of the filtrate to form anultrafiltrate, and purification of the enzyme from the ultrafiltrate,preferably by conventional liquid chromatography. The presentinvention 1) provides a method of large-scale (>20 million unit)production of reverse transcriptases, including MMLV-RT and mutants orderivatives thereof; and 2) provides a scalable method for theproduction of any desirable quantity of the desired enzyme.

[0018] The present methods are based in particular upon an optimizedmethod of chemical permeabilization of cells (preferably bacterialcells), which preferably strips the cell wall and yields spheroplasts(although conditions to merely permeabilize the cell wall to formprotoplasts may equally be used), and an optimized method of filtrationof the spheroplasts or protoplasts under conditions favoring the releaseof reverse transcriptase enzymes, but inhibiting the release of nucleicacids, from the spheroplasts or protoplasts. The permeabilizationprocess has been optimized to allow intracellular enzymes, particularlyreverse transcriptases, to permeate or cross the spheroplast orprotoplast membrane while preventing the cellular nucleic acids (DNAand/or RNA) from entering the permeation buffer. This approach providesan initial extract that is enriched in enzyme and that is relativelyfree of nucleic acids. The extract is then subjected to filtration underconditions (including precise definition of the variables of salt, pH,and choice of membrane chemistry) favoring release of the enzyme fromthe spheroplasts or protoplasts while preventing cells, cell debrisand/or nucleic acids from crossing the filtration membrane barriers.Following filtration (which may include microfiltration and/orultrafiltration), reverse transcriptase enzymes may be purified orisolated by standard techniques such as chromatography orelectrophoresis, to provide enzyme preparations of the invention.

[0019] Sources of Reverse Transcriptase Enzymes

[0020] Any reverse transcriptase enzymes may be prepared according tothe methods of the present invention from a variety of prokaryotic andeukaryotic cells including bacteria that are commercially available (forexample, from American Type Culture Collection (ATCC), Rockville, Md.and the Collection, Agricultural Research Culture Collection (NRRL),Peoria, Ill.). Examples of bacterial deposits as sources of RTs includeATCC deposit no. 67007 (M-MLV RT H+), ATCC deposit no. 67555 (M-MLV H−),NRRL B-21790 (AMV RT αH+/βH−), and NRRL B-21679 (RSV RT αH+/βH−).

[0021] Enzymes prepared in accordance with the invention include anyenzyme having reverse transcriptase activity. Such enzymes include, butare not limited to, retroviral reverse transcriptase, retrotransposonreverse transcriptase, hepatitis B reverse transcriptase, cauliflowermosaic virus reverse transcriptase, bacterial reverse transcriptase, andmutants, fragments, variants or derivatives thereof (see WO 98/47912,U.S. Pat. Nos. 5,668,005, and 5,017,492). As will be understood by oneof ordinary skill in the art, modified reverse transcriptases may beobtained by recombinant or genetic engineering techniques that areroutine and well-known in the art. Mutant reverse transcriptases can,for example, be obtained by mutating the gene or genes encoding thereverse transcriptase of interest by site-directed or randommutagenesis. Such mutations may include point mutations, deletionmutations and insertional mutations. Preferably, one or more pointmutations (e.g., substitution of one or more amino acids with one ormore different amino acids) are used to construct mutant reversetranscriptases of the invention. Fragments of reverse transcriptases maybe obtained by deletion mutation by recombinant techniques that areroutine and well-known in the art, or by enzymatic digestion of thereverse transcriptase(s) of interest using any of a number of well-knownproteolytic enzymes.

[0022] Preferred enzymes which may be prepared according to theinvention include those that are reduced or substantially reduced inRNase H activity. Such enzymes that are reduced or substantially reducedin RNase H activity may be obtained by mutating the RNase H domainwithin the reverse transcriptase of interest, preferably by one or morepoint mutations, one or more deletion mutations, and/or one or moreinsertion mutations as described above. By an enzyme “substantiallyreduced in RNase H activity” is meant that the enzyme has less thanabout 30%, less than about 25%, less than about 20%, more preferablyless than about 15%, less than about 10%, less than about 7.5%, or lessthan about 5%, and most preferably less than about 5% or less than about2%, of the RNase H activity of the corresponding wild type or RNase H+enzyme such as wild type Moloney Murine Leukemia Virus (M-MLV), AvianMyeloblastosis Virus (AMV) or Rous Sarcoma Virus (RSV) reversetranscriptases. The RNase H activity of any enzyme may be determined bya variety of assays, such as those described, for sample, in U.S. Pat.No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265(1988), in Gerard, G. F., et al., FOCUS 14(5):91 (1992), in WO 98/47912,and in U.S. Pat. No. 5,668,005, the disclosures of all of which arefully incorporated herein by reference.

[0023] Particularly preferred enzymes for use in the invention include,but are not limited to M-MLV H− reverse transcriptase, RSV H− reversetranscriptase, AMV H− reverse transcriptase, RAV H− reversetranscriptase, MAV reverse transcriptase and HIV H− reversetranscriptase (see WO 98/47912). It will be understood by one ofordinary skill, however, that any enzyme capable of producing a DNAmolecule from a ribonucleic acid molecule (i.e., having reversetranscriptase activity) that is reduced or not reduced in RNase Hactivity may be equivalently prepared in accordance with the invention.

[0024] It will be understood by one of ordinary skill in the art,however, that any cell, virus, microorganism or bacteria (includingprokaryotic and eukaryotic) may be used as a source for preparation ofreverse transcriptase enzymes (e.g., cellular source of RT) according tothe methods of the present invention. Preferably, recombinant cells(prokaryotic or eukaryotic) are used as a source of the reversetranscriptases in the methods of the invention. Such recombinant cellsmay be prepared by recombinant DNA techniques that are familiar to oneor ordinary skill in the art (see e.g., Kotewicz, M. L., et al., Nucl.Acids Res. 16:265 (1988); Soltis, D. A., and Skalka, A. M., Proc. Natl.Acad. Sci. USA 85:3372-3376 (1988)). Such sources of reversetranscriptases may be grown according to standard microbiologicaltechniques, using culture media and incubation conditions suitable forgrowing active cultures of the particular species that are well-known toone of ordinary skill in the art (see, e.g., Brock, T. D., and Freeze,H., J. Bacteriol. 98(1):289-297 (1969); Oshima, T., and Imahori, K.,Int. J. Syst. Bacteriol. 24(1):102-112 (1974)).

[0025] Permeabilization of Cells

[0026] In the initial steps of the present methods, a cellular source ofreverse transcriptase is treated under conditions to allow the releaseof the reverse transcriptase from the cell and preferably to retainnucleic acids in the cell. Such conditions may include permeabilizingthe cells by stripping away the cell walls and converting the cells intospheroplasts or permeabilizing the cell (making openings in the cellwall without totally removing it) to convert the cells into protoplasts.Such conditions may include chemical and/or enzymatic (e.g., lysozyme)treatment, although a variety of other techniques may be used for thispermeabilization. The production of substantially nucleic acid-freeenzymes by the present invention preferably uses a permeabilizationmethod which will produce protoplast or spheroplasts that retainsubstantially all the nucleic acids within the spheroplast orprotoplasts while allowing intracellular proteins (including enzymes) tomove across the spheroplast or protoplast membrane. All procedures frompermeabilization to final purification of the enzymes should be carriedout at temperatures below normal room temperature, preferably at about1-10° C., more preferably at about 2-8° C., and most preferably at about2-6° C., to prevent enzyme denaturation and loss of activity.Furthermore, all materials used throughout the present methods (i.e.,reagents, salts, chromatography resins, equipment) should be sterilizedby heat or barrier sterilization techniques (as appropriate to thematerial to be sterilized), to prevent the contamination of the reversetranscriptase enzymes with nucleic acids or other unwanted contaminants.

[0027] This permeabilization is preferably accomplished by suspension ofthe cells in an aqueous solution comprising at least one or morechaeotropic agents and/or nonionic detergent. According to a preferredembodiment, this permeabilization is preferably accomplished bysuspension of the cells in an aqueous solution comprising at least twononionic detergents. Chaeotropic agents preferable for use in themethods of the present invention include salts of guanidine or urea,most preferably guanidine hydrochloride. Any nonionic detergent may beused; most preferable are octylphenoxy-polyethoxyethanol nonionicsurfactant (TRITON X-100®), Brij 35, Tween 20 and Nonidet P-40 (NP-40®),although other nonionic surfactants and mixtures thereof, such asN-alkylglucosides, N-alkylmaltosides, glucamides, digitonin,deoxycholate,3-[3-cholamidopropyl)dimethyl-ammonium]-1-propane-sulfonate (CHAPS) orcetyltrimethyl-ammonium-bromide (CTAB) may also be used in the presentcompositions. Reagents such as chaeotropes, detergents, buffer salts,etc., are available commercially, for example from Sigma Chemical Co.(St. Louis, Mo.).

[0028] For permeabilization, the cells are preferably suspended in abuffered salt solution containing the chaeotrope(s) and/or thedetergent(s). Preferably, the solution is an aqueous solution with adistilled, deionized water (dH₂O) base consisting ofbis-trishydroxymethylaminomethane (BisTRIS® base) at a concentration ofabout 25-500 mM, preferably about 50-250 mM, more preferably about50-150 mM, and most preferably about 100 mM, at a pH of about 7.0-9.0,preferably about 7.0-8.5, more preferably about 7.0-8.0, more preferablyabout 7.0-7.5 and most preferably about 7.0 (pH at about 20-25° C.). Theconcentration of the chaeotrope in the solution is preferably about300-1000 mM, more preferably about 500-750 mM, and most preferably about600 mM. The concentration of the nonionic detergent is preferably about1-10% (vol/vol), more preferably about 2-8% and most preferably about2-5%. Within the context of the present invention, one or morechaeotropic agents and/or nonionic detergents may be used within theconcentration ranges specified. The permeabilization buffer solution mayalso comprise other components, such as protease inhibitors (e.g.,phenylmethylsulfonylfluoride, added at a final concentration of about0.5 mM), reducing agents (e.g., β-mercaptoethanol or most preferablydithiothreitol at a final concentration of about 1 mM), and chelatingagents (e.g., disodium ethylenediaminetetraacetic acid (Na₂EDTA), mostpreferably at a concentration of about 10 mM); this buffer compositionis referred to hereinafter as “permeabilization buffer.” It will beunderstood by one of ordinary skill in the art, however, that othersuitable buffer compositions may be substituted with equivalent effectin the permeabilization process.

[0029] For permeabilization, the cells are preferably suspended inpermeabilization buffer at a concentration of about 50-1000 g (wetweight) of cells per liter of solution, preferably about 100-500 μL, andmost preferably about 250 g/L (cell density of about 1-5×10¹⁰cells/gram, preferably about 2-5×10¹⁰ cells/gram, and most preferablyabout 2.5×10¹⁰ cells/gram). The cell suspension is gently stirred,preferably via magnetic or impeller stirring, in such a way as toprevent shearing and rupture of the cells. After about 30-60 minutes,most preferably about 45 minutes, a protein-extracting salt is added tothe suspension to enhance the permeation of the intracellular enzymesacross the spheroplast or protoplast membranes. Although any salt may beused in the present invention (except salts of toxic metals such ascadmium or other heavy metals), preferred salts include sodium chloride,potassium acetate, sodium acetate, ammonium acetate, ammonium chloride,ammonium sulfate or potassium chloride, most preferably ammoniumsulfate. Salt is added to the suspension at a concentration of about100-500 mM, preferably about 200-400 mM, and most preferably about 300mM. The salt should be gradually added to the solution to provide foroptimal solubilization. Following addition of the salt, the solution ismixed for about an additional 30-60 minutes, most preferably about anadditional 45 minutes, during which time the bacterial cells areconverted into spheroplasts or protoplast and the intracellularproteins, including reverse transcriptase enzymes, begin to cross thespheroplast or protoplast membrane while cellular nucleic acids arepreferably retained within the spheroplast or protoplast.

[0030] Microfiltration, Concentration and Diafiltration

[0031] Following permeabilization of the cells, reverse transcriptasesare collected by subjecting the spheroplasts or protoplast to filtration(e.g., microfiltraiton) to separate the enzymes from the spheroplasts orprotoplast and remove particulate matter. In another aspect, thefiltrate may be subjected to concentration and/or diafiltration. Thepresent methods obviates the need for precipitation of nucleic acidsand/or the use of centrifugation techniques; this elimation ofcentrifugation facilitates the rapid production of reverse transcriptaseenzymes at any scale in a continuous or discontinuous fashion. Thegeneral methods of filtration (e.g., microfiltration), concentration anddiafiltration are generally well-known to one of ordinary skills, andwill result in the preparation of an enzyme ultrafiltrate (which ispreferably nucleic-acid free) suitable for purification andcharacterization of the enzymes.

[0032] Microfiltration is preferably carried out by collecting thespheroplast/or protoplast solution in permeabilization buffer (describedabove) and diafiltering the solution against a filtration buffer thorugha semi-permeable membrane, most preferably a hydrophilic dialysis,microfiltration or ultrafiltration membrane. The filtration bufferpreferably is a dH₂O-based soltion comprising: a) a buffer salt,preferably trishydroxymethylaminomethane (TRIS base) at a concentrationof about 25-500 mM, preferably about 50-250 mM, more preferably about50-150 mM, and most preferably about 100 mM, at a pH of about 7.0-9.0,preferably about 7.0-8.5, more preferably about 7.0-8.0, and mostpreferably about 8.0 (pH at 4° C.); and b) the protein-extracting saltwhich was added to the permeabilization buffer, which is preferablyammonium sulfate, at a concentration of about 100-500 mM, preferablyabout 200-400 mM, and most preferably about 300 mM. The filtrationbuffer solution may also comprise other components, such as proteaseinhibitors (e.g., phenylmethysulfonylfluoride, added at a finalconcentration of about 1.0 mM), reducing agents (e.g., β-mercaptoethanolor most preferably dithiothreitol at a final concentration of about 1mM), and chelating agents (e.g., disodium ethylenediaminetetraaceticacid (Na₂EDTA), most preferably at a concentration of about 10 mM; thisbuffer composition is referred to hereinafter as “filtration buffer.” Itwill be understood by one of ordinary skill in the art, however, thatother suitable buffer compositions may be substituted with equivalenteffect in the filtration process.

[0033] Preferable for use in microfiltration is a system allowingpermeation of intracellular enzymes through the membrane and into thefiltrate, leaving spheroplasts and/or protoplast (with the nucleic acidsretained therein) and particulate matter in the retentate. One suitablesystem providing such conditions is, for example, a hollow fibermicrofiltration system which is commercially available (Spectrum),although similar systems providing the same results will be known to oneof ordinary skill. Following microfiltration in this manner, thefiltrate contains the reverse transcriptase enzymes which aresubstantially free of nucleic acids such as DNA, as the DNA ispartitioned from the enzymes by being retained with the particulatematter. This filtrate may then be concentrated, for example by membraneconcentration through a semi-permeable membrane using a commerciallyavailable system (AG/Technology Corp.) or equivalent. The enzymes maythen be individually purified from the concentrate as described below;alternatively, the concentrate may be diafiltered as described aboveagainst a suitable buffer solution to place the enzymes into anappropriate chemical environment for purification, as described in moredetail in Example 2.

[0034] Purification and Characterization of Enzymes

[0035] Following concentration and/or diafiltration as described above,reverse transcriptase enzymes may be purified by a variety of proteinpurification techniques that are well-known to one of ordinary skill inthe art. Suitable techniques for purification include, but are notlimited, ammonium sulfate or ethanol precipitation, acid extraction,preparative gel electrophoresis, immunoadsorption, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, immunoaffinitychromatography, size exclusion chromatography, liquid chromatography(LC), high performance LC (HPLC), fast performance LC (FPLC),hydroxylapatite chromatography, lectin chromatography, and immobilizedmetal affinity chromatography (IMAC). Most preferably, the enzymes arepurified by a combination of liquid chromatographic techniques includingion exchange, affinity and size exclusion methods such as thosedescribed in Example 3, although alternative chromatographic solidsupports, mobile phases and associated methods may be equivalently usedand will be well-known to one of ordinary skill. The invention thusprovides for substantially pure reverse transcriptases. Substantiallypure a used herein refers to a preparation or sample which issubstantially free of contaminating components, proteins etc. which mayadversely affect the activity or performance of the RT in the use of theenzyme such as in amplification or synthesis.

[0036] Assays for Nucleic Acid Content

[0037] Purified reverse transcriptase enzymes made according to thepresent invention may be examined for nucleic acid content by a varietyof methods which are well-known to one of ordinary skill in the art. Forexample, a sample of the final product can be assayed by ultravioletspectrophotometry, comparing absorption of light by the sample at awavelength of 260 nm (A₂₆₀, the absorption maximum for DNA) to that at280 nm (A₂₈₀, the absorption maximum for tryptophan, which is found inmost proteins); the lower the A₂₆₀/A₂₈₀ ratio, the lower the content ofDNA in the sample. Samples with minimal A₂₆₀/A₂₈₀ values may then bepooled to constitute a substantially nucleic acid-free preparation ofreverse transcriptase enzymes.

[0038] Alternatively, samples may be directly assayed for the presenceof DNA or other nucleic acids by gel electrophoresis or dot blotting andstaining with a DNA-binding dye (e.g., ethidium bromide, acridineorange, Hoechst stain, pico green) or antibody, which are commerciallyavailable, for example, from Sigma (St. Louis, Mo.). In addition, theDNA content of samples of reverse transcriptases may be examined bycarrying out an amplification reaction in the absence of exogenouslyadded DNA template, either as a “notemplate control” in a standard PCRassay (Rand, K. H., and Houck, H., Mol. Cell Probes 4(6):445-450(1990)), or by specifically designing an assay to measure DNA content byradiolabeled nucleotide incorporation into salmon testes or bovinethymus DNA, according to methods that are standard in the art. Use ofsuch assays will allow one of ordinary skill, without undueexperimentation, to identify samples of reverse transcriptase enzymesobtained by the purification schemes described above, which may then bepooled and used as preparations of substantially nucleic acid-freereverse transcriptase enzymes.

[0039] Formulation of Enzymes

[0040] Following their purification or isolation, the substantially pureand preferably substantially DNA-free reverse transcriptase enzymes maybe stored until use in a buffered solution at temperatures of about−φ80° to 25° C., most preferably at −80° to 4° C., or in lyophilizedform. Alternately, the enzymes may be stabilized by drying in thepresence of a sugar such as trehalose (U.S. Pat. Nos. 5,098,893 and4,824,938) or acacia gum, pectin, carboxymethylcellulose,carboxymethyl-hydroxyethylcellulose, guar, carboxy guar,carboxymethylhydroxypropyl guar, laminaran, chitin, alginates orcarrageenan. In addition, the enzymes provided by the present inventionmay be directly formulated into compositions to be used in techniquesrequiring the use of reverse transcriptase enzymes, such as compositionsfor nucleic acid synthesis or amplification.

[0041] Kits

[0042] In other preferred embodiments, the substantially pure andpreferably substantially DNA-free reverse transcriptases provided by thepresent invention may be assembled into kits for use in methodsrequiring reverse transcriptase enzymes, such as nucleic acid synthesis(e.g., cDNA synthesis), amplification (e.g., RT-PCR) or sequencingutilizing RT. The kit according to the present invention comprises acarrier means having in close confinement therein one or more containermeans, such as vials, tubes, bottles and the like, wherein a firstcontainer means contains a reverse transcriptase of this invention. Thekit encompassed by this aspect of the present invention may furthercomprise in the same or different containers additional reagents andcompounds necessary for carrying out standard nucleic synthesis,amplification and sequencing protocols. Such additional components mayinclude reaction buffers, nucleotides (e.g., dTTP, dATP, dCTP, dGTP,ddATP, ddTTP, ddGTP, ddCTP and derivatives thereof including labelednucleotides), one or more DNA polymerases (such as Taq DNA polymerase),one or more primers and the like.

[0043] Use of the Reverse Transcriptase Enzymes

[0044] The substantially pure or substantially DNA-free reversetranscriptase enzymes and kits embodied in the present invention willhave general utility in any application utilizing reverse transcriptaseenzymes, including but not limited to nucleic acid cDNA synthesis, andnucleic acid amplification or sequencing methodologies.

[0045] In a first aspect, the RTs of the invention may be used forsynthesis of nucleic acid molecules. Such methods for making one or morenucleic acid molecules, comprising mixing one or more nucleic acidtemplates (preferably one or more RNA templates and most preferably oneor more messenger RNA templates) with one or more polypeptides havingreverse transcriptase activity and incubating the mixture underconditions sufficient to make a first nucleic acid molecule or moleculescomplementary to all or a portion of the one or more nucleic acidtemplates. In a preferred embodiment, the first nucleic acid molecule isa single-stranded cDNA. Nucleic acid templates suitable for reversetranscription according to this aspect of the invention include anynucleic acid molecule or population of nucleic acid molecules(preferably RNA and most preferably mRNA), particularly those derivedfrom a cell or tissue. In a preferred aspect, a population of mRNAmolecules (a number of different mRNA molecules, typically obtained fromcells or tissue) are used to make a cDNA library, in accordance with theinvention. Preferred cellular sources of nucleic acid templates includebacterial cells, fungal cells, plant cells and animal cells.

[0046] RT enzymes made in accordance with the invention may also be usedin methods for amplifying and sequencing nucleic acid molecules. Nucleicacid amplification methods according to this aspect of the invention maybe one step (e.g., one-step RT-PCR) or two-step (e.g., two-step RT-PCR)reactions. According to the invention, one-step RT-PCR type reactionsmay be accomplished in one tube thereby lowering the possibility ofcontamination. Such one-step reaction comprise (a) mixing a nucleic acidtemplate (e.g., mRNA) with one or more polypeptides having reversetranscriptase activity and with one or more DNA polymerases and (b)incubating the mixture under conditions sufficient to amplify a nucleicacid molecule complementary to all or a portion of the template with oneor more polypeptides having reverse transcriptase activity (andoptionally having DNA polymerase activity). Incubating such a reactionmixture under appropriate conditions allows amplification of a nucleicacid molecule complementary to all or a portion of the template. Suchamplification may be accomplished by the reverse transcriptase activityalone or in combination with the DNA polymerase activity. Two-stepRT-PCR reactions may be accomplished in two separate steps. Such amethod comprises (a) mixing a nucleic acid template (e.g., mRNA) withone or more reverse transcriptases, (b) incubating the mixture underconditions sufficient to make a nucleic acid molecule (e.g., a DNAmolecule) complementary to all or a portion of the template, (c) mixingthe nucleic acid molecule with one or snore DNA polymerases and (d)incubating the mixture of step (c) under conditions sufficient toamplify the nucleic acid molecule. For amplification of long nucleicacid molecules (i.e., greater than about 3-5 Kb in length), acombination of DNA polymerases may be used, such as one DNA polymerasehaving 3′ exonuclease activity and another DNA polymerase beingsubstantially reduced in 3′ exonuclease activity. An alternativetwo-step procedure comprises the use of one or more polypeptides havingreverse transcriptase activity and DNA polymerase activity (e.g., Tth,Tma or Tne DNA polymerases and the like) rather than separate additionof a reverse transcriptase and a DNA polymerase.

[0047] Nucleic acid sequencing methods according to this aspect of theinvention may comprise both cycle sequencing (sequencing in combinationwith amplification) and standard sequencing reactions. The sequencingmethod of the invention thus comprises (a) mixing a nucleic acidmolecule to be sequenced with one or more primers, two or more reversetranscriptases, one or more nucleotides and one or more terminatingagents, (b) incubating the mixture under conditions sufficient tosynthesize a population of nucleic acid molecules complementary to allor a portion of the molecule to be sequenced, and (c) separating thepopulation to determine the nucleotide sequence of all or a portion ofthe molecule to be sequenced. According to the invention, one or moreDNA polymerases (preferably thermostable DNA polymerases) may be used incombination with or separate from the reverse transcriptases.

[0048] Amplification methods in which the present enzymes may be usedinclude PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202), StrandDisplacement Amplification (SDA; U.S. Pat. No. 5,455,166; EP 0 684 315),and Nucleic Acid Sequence-Based Amplification (NASBA; U.S. Pat. No.5,409,818; EP 0 329 822). Nucleic acid sequencing techniques which mayemploy the present enzymes include dideoxy sequencing methods such asthose disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523, as well asmore complex PCR-based nucleic acid fingerprinting techniques such asRandom Amplified Polymorphic DNA (RAPD) analysis (Williams, J. G. K., etal., Nucl. Acids Res. 18(22):6531-6535, 1990), Arbitrarily Primed PCR(AP-PCR; Welsh, J., and McClelland, M., Nucl. Acids Res.18(24):7213-7218, 1990), DNA Amplification Fingerprinting (DAF;Caetano-Anolles et al., Bio/Technology 9:553-557, 1991) microsatellitePCR or Directed Amplification of Minisatellite-region DNA (DAMD; Heath,D. D., et al., Nucl. Acids Res. 21(24):5782-5785, 1993), andAmplification Fragment Length Polymorphism (AFLP) analysis (EP 0 534858; Vos, P., et al., Nucl. Acids Res. 23 (21):4407-4414, 1995; Lin, J.J., and Kuo, J., FOCUS 17(2):66-70, 1995). In particular, the enzymesand kits of the present invention will be useful in the fields ofmedical therapeutics and diagnostics, forensics, and agricultural andother biological sciences, in any procedure utilizing reversetranscriptase enzymes.

[0049] It will be readily apparent to one of ordinary skill in therelevant arts that other suitable modifications and adaptations to themethods and applications described herein are obvious and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1 Permeabilization of Bacterial Cells

[0050] In the initial steps of the purification process, 20 kg bacterialcells (E. coli, N4830 (pRT601) (see U.S. Pat. No. 5,017,492; ATCCdeposit no. 67007) containing the expression vector for MMLV-RT whichwere obtained directly from actively growing cultures were suspended at250 g of cells/L into cold (4° C.) permeabilization buffer. (100 mMBisTRIS, 5.0% Triton X-100, 2.0% sodium deoxycholic acid, 10 mM EDTA, 1mM dithiothreitol (DTT), pH 7.0.

[0051] During suspension of the cells in the buffer,phenylmethylsulfonylfluoride (PMSF) was added to a final concentrationof 1.0 mM. Cells were stirred for about 45 minutes at 4° C. to ensurecomplete suspension, and then ammonium sulfate was added to a finalconcentration of 300 mM and the cell suspension was stirred for anadditional 45 minutes. During this time, cells were permeabilized viathe action of the deoxycholic acid and Triton X-100, and intracellularprotein release into the buffer was enhanced by the action of theammonium sulfate.

Example 2 Microfiltration, Concentration and Diafiltration of Extracts

[0052] Microfiltration of the suspension was then carried out through120 ft² 0.2 μm Microgon mixed ester cellulose hollow fiber system, usinga recirculation rate of 120 L/min. The suspension was diafiltered withfive to six volumes of cold filtration buffer, collecting the permeatein a suitable sized chilled (4° C.) container. Under these conditions,recombinant enzymes passed through the membrane with the permeate,leaving the bacterial cells in the retentate.

[0053] As the ultrafiltration proceeded, concentration of the permeatewas begun once a sufficient volume had been collected to prime thesecond ultrafiltration system. Permeate was concentrated using an AmiconDC-90 system, through an AG technologies 10,000 MWCO membrane (althoughalternative membrane systems of 10,000 MWCO, such as a Filtron system, aMillipore plate and frame system, or a membrane from Microgon may bealso used) and an in-line chiller to minimize heat build-up from thepumping system. Permeate was concentrated to approximately the originalvolume of the extract (see Example 1), and was then diafiltered againstabout seven volumes of diafiltration buffer (20 mM NaPi, 100 mM NaCl,10.0 mM EDTA, 1 mM DTT, pH 6.5), until the conductivity was <7 mS.Ultrafiltrate was then immediately used for purification of the enzyme(Example 3).

Example 3 Purification and Characterization of DNA-Free Enzyme

[0054] Purification of the enzyme from the ultrafiltrate wasaccomplished by a series of chromatographic steps, using a proceduremodified slightly from that described for purification of T5 DNApolymerase from E. coli (Hughes, A. J., Jr., et al., J. Cell Biochem.Suppl. 0 16 (Part B):84 (1992)).

[0055] A. Macroprep High S

[0056] The filtrate was mixed with 9L Whatman DE-52 and then was polishfiltered through two CUNO 8ZP 10 A depth filters. In the firstchromatographic step, the ultrafiltrate was applied to a 9L BioRadMacroprep High S. The column was then washed with 10 volumes of 20 mMTRIS, 150 mM NaCl, 0.1 mM EDTA, 10% glycerol, 0.01% Triton X-100, 1 mMDTT, pH 8.0 at 4.0° C. run at a flow rate of about 20 cm/hr. Productelution was effected with a ten column volume gradient of the washbuffer to this same buffer containing 800 mM NaCl w/o EDTA run at 10cm/hr. Fractions demonstrating at least ⅓ of the large UV peak werepooled and subjected to further purification.

[0057] B. Macroprep Ceramic HTP

[0058] Pooled eluate from the High S Column was applied at a flow-rateof 20 cm/hr to a 6 L column of Macroprep Ceramic HTP, and the column wasthen washed with 5 volumes of 20 mM potassium phosphate, 100 mM KCl, 10%glycerol, 0.01% Triton X-100, 1 mM DTT, pH 7.0 at 4° C. at a flow-rateof 10 cm/hr. Fractions showing at least ⅕ of the UV peak were pooled andsubjected to further purification.

[0059] C. Fractogel COO-Column

[0060] The pool from the Ceramic HTP column was diluted with an equalvolume of 100 mM TRIS, 100 mM NaCl, 0.2 mM EDTA, 30% glycerol, 0.01%Triton X-100, 1 mM DTT, pH 7.5 at 4.0° C. and applied at a flow rate of20 cm/hr to a Fractogel COO-column (E. Merck, Inc.), which concentratesthe product. The column was then washed with 2 column volumes of lowsalt Fractogel COO-buffer (20 mM TRIS, 100 mM NaCl, 20% glycerol, 0.1 mMEDTA, 0.01% Triton X-100, 1 mM DTT, pH 7.5), and the enzyme eluted with50% high salt ceramic Fractogel COO-buffer (20 mM TRIS, 400 mM NaCl, 0.1mM EDTA, 20% glycerol, 0.01% Triton X-100, 1.0 mM DTT, pH 7.5 at 4.0° C.at 20 cm/hr, and fractions containing the UV peak were collected, andpooled as described above.

[0061] D. Dialysis

[0062] Fractogel COO-pool was dialyzed against 20 volumes of dialysisbuffer (20 mM TRIS, 0.1 mM EDTA, 50% (vol/vol) glycerol, 100 mM NaCl,0.01% Triton X-100, 1 mM DTT, pH 7.5) for 24 hours. Purified enzyme bulkwas then stored at −20° C. until used.

[0063] E. DNA Contamination Assays

[0064] To determine the extent of DNA contamination of variouspreparations of RT, samples of RT obtained from commercial sources maybe compared to a preparation made according to the methods of thepresent invention in a polymerase assay similar to that outlined above,except that no salmon testes DNA template is included in the reactionmixture. Briefly, reaction mixtures (500 μl) containing 25 mM TAPS (pH9.3), 10 mM MgCl₂, 50 mM KCl, 1 mM DTT, 100 μM each of dATP, dTTP, dGTPand dCTP, and 600 cpm of [³H]dTTP/pmol of total nucleotide are preparedand pre-incubated at 72° C. for five minutes. 100 units of M-MLV RT areadded to the reaction mixtures and then 100 units of purified DNA-freeTaq DNA polymerase (see U.S. Pat. No. 5,861,295) are added at specifictime intervals to initiate the reaction. A 30 μl sample is removed andadded to a vial containing 5 μl of 500 mM EDTA on ice. Once all timepoints are collected, a 20 μl aliquot of the quenched reaction sample isapplied to a GF/C filter, which is washed, dried and counted asdescribed above. Results are expressed as ³H incorporation (cpm) at eachtime point.

[0065] Other commercially available preparations of RT may be comparedto the preparations provided by the present invention for their DNAcontent. Together, the results should indicate that preparations of RTprovided by the present invention are substantially free of nucleicacids, while several commonly used commercial preparations of RT containsubstantial amounts of contaminating DNA.

[0066] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those of ordinary skill in theart that the same can be performed by modifying or changing theinvention within a wide and equivalent range of conditions, formulationsand other parameters without affecting the scope of the invention or anyspecific embodiment thereof, and that such modifications or changes areintended to be encompassed within the scope of the appended claims.

[0067] All publications, patents and patent applications cited hereinare indicative of the level of skill of those skilled in the art towhich this invention pertains, and are herein incorporated by referencein their entirety.

What is claimed is:
 1. A method of isolating or purifying a reversetranscriptase, said method comprising permeabilizing a cellular sourceof reverse transcriptase, subjecting said permeabilized cellular sourceof reverse transcriptase to filtration, and isolating said reversetranscriptase.
 2. The method of claim 1, wherein said reversetranscriptase is substantially free of nucleic acids.
 3. The method ofclaim 1, wherein said cellular source is a bacterial cell or arecombinant bacterial cell.
 4. The method of claim 3, wherein saidpermeabilization forms spheroplasts and/or protoplasts.
 5. The method ofclaim 4, wherein said filtration comprises microfiltration and/orultrafiltration.
 6. The method of claim 1, wherein said permeabilizationis accomplished by contacting said cellular source with an aqueoussolution comprising at least one component selected from the groupconsisting of a chaeotropic agent and/or a nonionic detergent.
 7. Themethod of claim 6, wherein said nonionic detergent is selected from thegroup consisting of Triton X-100 and sodium deoxycholic acid.
 8. Themethod of claim 1, wherein said isolation step comprises columnchromatography.
 9. The method of claim 1, wherein said method isconducted under conditions favoring the partitioning of nucleic acidsfrom said reverse transcriptase.
 10. The method of claim 9, wherein saidconditions comprise microfiltration of spheroplast or protoplasts in thepresence of ammonium sulfate.
 11. The method of claim 1, wherein saidreverse transcriptase is M-MLV RT or M-MLV RT substantially reduced inRNase H activity.
 12. A reverse transcriptase made according to themethod of claim
 1. 13. The reverse transcriptase of claim 12, whereinsaid reverse transcriptase is selected from the group consisting ofM-MLV RT or M-MLV RT substantially reduced in RNase H activity.
 14. Akit for synthesizing a nucleic acid molecule comprising a reversetranscriptase made according to the method of claim
 1. 15. The kit ofclaim 14, wherein said kit further comprises at least one componentselected from the group consisting of one or more DNA polymerases, oneor more nucleotides, one or more buffers, and one or more primers. 16.The kit of claim 15, wherein said kit may be used for RT PCR.